CD20 single-domain antibody and its humanized antibody F11

Abstract

This application relates to the field of biomedicine, specifically to CD20 single-domain antibodies and their humanized antibody F11. Specifically, this application provides a CD20 antibody that specifically binds to CD20. The antibody contains three complementarity-determining regions CDR1, CDR2, and CDR3, and the complementarity-determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 1-3 in sequence. Further, based on this antibody, an immune cell modified with a dual chimeric antigen receptor gene by tandem CD19 antibody is constructed and its application is provided. In particular, a CAR-T cell for cell therapy is provided.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and specifically relates to a CD20 single-domain antibody and its humanized antibody F11. Background Art

[0002] B cells play an important role in the humoral immune response and antigen presentation of the human body. The consequences of B cell lesions are very serious, and the diseases caused by them are mainly two categories. The first category is malignant B cell blood cancers, and the second category is autoimmune diseases caused by abnormal B cells recognizing self-antigens. CD20 plays an important role in the immune response process of B cells. It appears from the late pro-B stage, and its expression level gradually increases with the maturation of B cells. CD19 is a surface protein expressed on B lymphocytes and follicular dendritic cells and belongs to a member of the immunoglobulin (Ig) superfamily. CD19 plays an important role in the development of early B cells, and all B cell acute leukemias express CD19.

[0003] In CAR-T (chimeric antigen receptor T cell) therapy, these two antigens have become key targets for treating certain types of B cell malignancies (such as non-Hodgkin lymphoma and chronic lymphocytic leukemia).

[0004] CAR is an artificial recombinant protein mainly composed of an antigen recognition region, an extracellular hinge region, a transmembrane region, and an intracellular signal transduction domain. The extracellular domain is the antigen-targeting part purified from monoclonal antibodies. After binding to tumor antigens, it triggers cell activation, mediates cytokine release, cell lysis degranulation, and T cell proliferation. It determines the quality, intensity, and persistence of the T cell response to tumor antigens. The transmembrane region is mainly used to maintain the CAR structure, and the T cell receptor constitutes the intracellular signal transduction domain of the signal pathway required to activate T cells. After CAR-T expansion, it can produce a strong immune response effect to attack and destroy tumors. Summary of the Invention

[0005] Aiming at the situation that the long-term effect of single targeting in current CAR technology for treating tumors is not very ideal, and the tumor microenvironment affects the treatment effect of CAR technology, this application provides a new CD20 antibody, and constructs a dual chimeric antigen receptor gene-modified immune cell and its application based on the tandem CD19 antibody of this antibody. In particular, it provides a CAR-T cell for cell therapy.

[0006] The technical solution adopted in this application is:

[0007] In a first aspect, the present application provides a CD20 antibody that specifically binds to CD20. The antibody comprises three complementarity-determining regions CDR1, CDR2, and CDR3. The amino acid sequence of the complementarity-determining region CDR1 of the antibody is as shown in SEQ ID NO.1, the amino acid sequence of the complementarity-determining region CDR2 is as shown in SEQ ID NO.2, and the amino acid sequence of the complementarity-determining region CDR3 is as shown in SEQ ID NO.3.

[0008] Preferably, the CD20 antibody includes antigen-binding fragments such as single-domain antibodies, monoclonal antibodies, single-chain antibodies, Fab, Fab’, Fv fragments, F(ab’)2, scFv, di-scFv, etc.

[0009] Preferably, the CD20 antibody is a single-domain antibody.

[0010] The single-domain antibody of the present invention can also be referred to as a "nanobody (Nb)", "VHH antibody (variable domain of heavy chain of heavy-chain antibody, VHH antibody)", or "single-domain heavy-chain antibody (single domain antibody, sdAb)", and refers to a genetically engineered antibody composed only of the variable region of a heavy-chain antibody.

[0011] Furthermore, the CD20 antibody further includes a functionally conservative variant or a functional variant of the CD20 antibody.

[0012] Specifically, the functionally conservative variant of the antibody includes the replacement of amino acids in the form of polarity, hydrogen bond potential, acidity, alkalinity, hydrophobicity, aromatic groups, etc. The "functionally conservative variant" refers to a variant in which the replacement is made according to a given amino acid in the antibody of the present application without compromising the overall conformation and function of the antibody, including the replacement of one amino acid with another having similar properties (e.g., polarity, hydrogen bond potential, acidity, alkalinity, presence of aromatic groups, etc.). Amino acids with similar properties are well known to those skilled in the art.

[0013] Specifically, the functional variants include, but are not limited to: derivatives that are substantially similar in primary structure sequence but include modifications that the parental antibody of the present application does not have, such as chemical and / or biochemical modifications in vitro or in vivo. These modifications include, for example, acetylation, acylation, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, cross-linking, formation of disulfide bonds, glycosylation, hydroxylation, methylation, oxidation, polyethylene glycolylation, proteolytic treatment, phosphorylation. Such functional variants are also included within the scope of protection of the present application.

[0014] Preferably, the antibody is humanized. The immunogenicity of a humanized antibody should theoretically be less than that of a chimeric antibody, making it more suitable for large-scale and repeated use.

[0015] Preferably, the CD20 antibody has an amino acid sequence shown in any one of SEQ ID NOs. 4-9.

[0016] Preferably, the CD20 antibody further includes a single-domain antibody corresponding to an amino acid sequence or nucleotide sequence having at least 75% identity with the amino acid sequences shown in SEQ ID NOs. 4-9, that is, an amino acid sequence or nucleotide sequence obtained by replacing one or more amino acids or nucleotides at any position with other arbitrary amino acids or nucleotides on the basis of the corresponding amino acid sequence or nucleotide sequence, as long as the amino acid sequence or nucleotide sequence having at least 75% identity with the above-mentioned amino acid sequence or nucleotide sequence of the present application is within the protection scope of the present application.

[0017] In a second aspect, the present application provides a humanized CD19 antibody, the CD19 antibody comprising VH and VL, the amino acid sequence of the VL being as shown in SEQ ID NO. 24, and the amino acid sequence of the VH being as shown in SEQ ID NO. 28; or, the amino acid sequence of the VL being as shown in SEQ ID NO. 25, and the amino acid sequence of the VH being as shown in SEQ ID NO. 29.

[0018] Preferably, the VH and VL are connected by a linker.

[0019] In a third aspect, the present application provides a fusion protein, the fusion protein containing the CD20 antibody described in the first aspect of the present application and the CD19 antibody described in the second aspect of the present application.

[0020] More specifically, the fusion protein from the N-terminus to the C-terminus is in turn: the aforementioned CD20, the first linker, the light chain variable region of the aforementioned CD19 antibody, the second linker, and the heavy chain variable region of the aforementioned CD19 antibody.

[0021] The first linker and the second linker may be the same or different.

[0022] The linker (also known as the fusion protein linker, linker) described in the present application includes (GGGGS)n, (GGGS)n, (SSSSG)n, (GSGSA)n, and (GGSGG)n, etc. Preferably, the linker may further include other amino acids.

[0023] Fourth aspect, the present application provides a chimeric antigen receptor, which comprises any one of the following: the CD20 antibody described in the first aspect, the CD19 antibody described in the second aspect of the present application, or the fusion protein described in the third aspect of the present application.

[0024] Preferably, the chimeric antigen receptor further comprises a hinge region. More preferably, the hinge region includes the hinge regions of the following molecules: CD8α, CD28, CD34, 4-1BB, OX40, CD3ε, IgG1, IgG4, PD-1, IL-2 receptor, IL-7 receptor, IL-11 receptor. Most preferably, the hinge region is the CD8α hinge region.

[0025] Preferably, the chimeric antigen receptor further comprises a transmembrane domain. Preferably, the transmembrane domain includes the transmembrane domains of the following molecules: CD8α, CD28, 4-1BB, CD34, CD3ε, PD-1, IgG1, IgG4, OX40, IL-2 receptor, IL-7 receptor, IL-11 receptor. Most preferably, the transmembrane domain is the CD8α transmembrane domain.

[0026] Preferably, the chimeric antigen receptor further comprises a co-stimulatory signal domain. Preferably, the co-stimulatory signal domain includes the co-stimulatory signal domains of the following molecules: 4-1BB (CD137), CD19, CD4, CD27, CD28, ICOS (CD278), CD8α, CD8β, BAFFR, HVEM, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1, B7-H3, OX40, DR3, GITR, CD30, TIM1, CD2, CD7, CD226. Most preferably, the co-stimulatory signal domain is the 41BB co-stimulatory signal domain.

[0027] Preferably, the chimeric antigen receptor further comprises an intracellular signaling domain. Preferably, the intracellular signaling domain includes the intracellular signaling domains of the following molecules: CD3ζ, CD3γ, ZAP70, CD3δ, CD3ε, FcRγ, FcRβ, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, CD66d. Most preferably, the intracellular signaling domain is the CD3ζ (CD3z) intracellular signaling domain.

[0028] Preferably, the chimeric antigen receptor, from the N-terminus to the C-terminus, is: the CD20 antibody described in the first aspect / the CD19 antibody described in the second aspect of the present application / the fusion protein described in the third aspect of the present application - CD8 hinge region - CD8 transmembrane domain - 41BB co-stimulatory domain - CD3ζ intracellular signaling domain.

[0029] The term "chimeric antigen receptor (CAR)" generally refers to a fusion protein that includes an extracellular domain capable of binding an antigen and at least one intracellular domain. CAR is the core component of chimeric antigen receptor T cells (CAR-T), and it can include an antigen (e.g., tumor-specific antigen and / or tumor-associated antigen) binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain. The CAR of the present application may include linker connections between various domains added for proper spacing and conformation of the molecule. CAR is an engineered receptor that can implant any specific receptor onto immune effector cells, and in the present application, it especially refers to T cells, that is, prepared into CAR-T cells.

[0030] On the other hand, the present application provides a nucleic acid molecule that encodes one or more of the following: the CD20 antibody, the CD19 antibody, the fusion protein, and the chimeric antigen receptor described above in the present application.

[0031] On the other hand, the present application provides a recombinant vector that contains the aforementioned nucleic acid molecule.

[0032] Preferably, the vector includes a DNA vector, an RNA vector, a plasmid, and a virus-derived vector.

[0033] More preferably, the virus-derived vector includes a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a poxviral vector, and a herpesviral vector.

[0034] In certain embodiments, the present application has no particular limitation on the vector, and its selection depends on the desired function. Non-limiting examples of vectors include plasmid vectors, virus-derived vectors, phage vectors, and other vectors commonly used in, for example, genetic engineering. Various plasmids and vectors can be constructed based on methods well known to those skilled in the art.

[0035] In another aspect, the present application provides an engineered cell that contains or expresses the aforementioned CD20 antibody, CD19 antibody, fusion protein, chimeric antigen receptor, nucleic acid molecule, and recombinant vector;

[0036] Alternatively, the cell is prepared from the aforementioned recombinant vector. For example, the vector described in the present application can be introduced into the cell, and the vector described in the present application can be introduced into the cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.

[0037] Preferably, the cell is a human cell.

[0038] More preferably, the cell is an immune cell.

[0039] Preferably, the immune cells include T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages, gdT cells, any immune cells derived from iPS, or any combination thereof.

[0040] Preferably, the immune cell is a T cell.

[0041] Most preferably, the cell is a CAR-T cell.

[0042] On the other hand, the present application provides an antibody-drug conjugate, which comprises the CD20 antibody or CD19 antibody described in the present application, and the CD20 antibody or CD19 antibody is conjugated to one or more drugs.

[0043] On the other hand, the present application provides a pharmaceutical composition, which contains one or more of the aforementioned CD20 antibody, CD19 antibody, fusion protein, chimeric antigen receptor, nucleic acid molecule, recombinant vector, cell, and antibody-drug conjugate.

[0044] Preferably, the pharmaceutical composition further contains a pharmaceutically or physiologically acceptable carrier and / or excipient.

[0045] In certain embodiments, the pharmaceutically or physiologically acceptable carrier and / or excipient may comprise a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). The dosage of the active ingredient is a therapeutically effective amount. In addition, one or more of the aforementioned CD20 antibody, CD19 antibody, fusion protein, chimeric antigen receptor, nucleic acid molecule, recombinant vector, cell, and antibody-drug conjugate described in the present application can also be used together with other therapeutic agents.

[0046] Preferably, the pharmaceutical composition contains T cells expressing the aforementioned chimeric antigen receptor.

[0047] In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g., 0.9% (w / v) NaCl), glucose solutions (e.g., 5% glucose), solutions containing surfactants (e.g., 0.01% polysorbate 20), pH buffer solutions (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0048] The pharmaceutical compositions provided by the present application can be made into various dosage forms as needed, and the clinician can determine the dose beneficial to the patient according to factors such as the type, age, weight, and general disease condition of the subject patient, and the administration method. The administration method can be, for example, injection or any other suitable administration method known to those skilled in the art.

[0049] On the other hand, the present application provides an antibody containing a label, and the antibody is the aforementioned CD19 antibody and / or CD20 antibody.

[0050] Preferably, the label includes a radionuclide, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, an enzyme, a photosensitizing diagnostic agent.

[0051] In certain embodiments, the antibody containing a label can be assembled into a kit for the detection of CD19 and / or CD20. The kit further includes a container, instructions for use, a buffer, etc. In other embodiments, the kit further contains a lysis medium for dissolving the sample to be tested, general reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

[0052] On the other hand, the present application provides a method for preventing and / or treating CD20-related cancers, which includes administering an effective amount of the aforementioned CD20 antibody, fusion protein, chimeric antigen receptor, nucleic acid molecule, recombinant vector, cell, antibody-drug conjugate, pharmaceutical composition to a subject in need thereof.

[0053] On the other hand, the present application provides a method for preventing and / or treating CD19-related cancers, which includes administering an effective amount of the aforementioned CD19 antibody, fusion protein, chimeric antigen receptor, nucleic acid molecule, recombinant vector, cell, antibody-drug conjugate, pharmaceutical composition to a subject in need thereof.

[0054] Specifically, the method is to administer a therapeutically effective amount of CAR-T cells, and the CAR has the CD20 antibody and / or CD19 antibody provided by the present application.

[0055] In the present application, the term "administer" generally refers to via any route known in the art, and specifically may include: intravenous, intramuscular, intradermal, subcutaneous, transdermal, mucosal, intratumoral or mucosal. Medicinal carriers and formulations or compositions are also well known in the art.

[0056] In the present application, the term "effective amount" or "effective dose" generally refers to an amount sufficient to achieve or at least partially achieve the desired effect. The "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is generally any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes the regression of a disease (as evidenced by a reduction in the severity of the disease symptoms, an increase in the frequency and duration of the asymptomatic period of the disease, or the prevention of damage or disability caused by the disease).

[0057] On the other hand, the present application provides a method for detecting CD20 protein or diagnosing CD20-related cancers, the method comprising contacting a sample with the aforementioned CD20 antibody and fusion protein of the present application and detecting the complex, wherein detecting the complex indicates the expression of CD20 protein in the sample.

[0058] On the other hand, the present application provides a method for detecting CD19 protein or diagnosing CD19-related cancers, the method comprising contacting a sample with the aforementioned CD19 antibody and fusion protein of the present application and detecting the complex, wherein detecting the complex indicates the expression of CD19 protein in the sample.

[0059] Preferably, the detection is performed in vitro on an ex vivo sample.

[0060] Preferably, the detection is for non-diagnostic purposes.

[0061] Preferably, the detection can be qualitative, quantitative, or semi-quantitative.

[0062] On the other hand, the present application provides the use of any one or more of the following products in detecting CD20, preparing diagnostic products and drugs for CD20-related cancers: the aforementioned CD20 antibody, fusion protein, chimeric antigen receptor, nucleic acid molecule, recombinant vector, cell, antibody-drug conjugate, pharmaceutical composition.

[0063] On the other hand, the present application provides the use of any one or more of the following products in detecting CD19, preparing diagnostic products and drugs for CD19-related cancers: the aforementioned CD19 antibody, fusion protein, chimeric antigen receptor, nucleic acid molecule, recombinant vector, cell, antibody-drug conjugate, pharmaceutical composition.

[0064] Preferably, the CD19-related cancers described in the present application include lymphoma and leukemia.

[0065] Preferably, the CD19-related cancer is leukemia.

[0066] Preferably, the CD19-related cancer is chronic myeloid leukemia.

[0067] Preferably, the CD20-related cancers described in this application include lymphoma and leukemia, specifically including non-Hodgkin lymphoma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, B-cell lymphoma, multiple sclerosis, rheumatoid arthritis, marginal zone lymphoma, small lymphocytic lymphoma, lupus nephritis, mantle cell lymphoma, systemic lupus erythematosus, pemphigus, immune thrombocytopenia, membranous nephropathy, B-cell blood cancer, acute lymphocytic leukemia, microscopic polyangiitis, kidney transplant rejection, and nephrotic syndrome.

[0068] Preferably, in the specific embodiments of this application, the cell line of chronic myeloid leukemia is used as the target cell for experimental verification.

[0069] The term "T cell" or "T lymphocyte" as used in this application can be any T cell, such as cultured T cells, primary T cells, or T cells obtained from mammals (preferably primates, species including monkeys, dogs, or humans). T cells can be any type of T cell, including NKT cells, and can be at any stage of development. If obtained from a mammal, then the T cells can be obtained from many sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. Description of the Drawings

[0070] Figure 1 It is a graph showing the binding affinity detection results of the F11 original antibody and the humanized antibody HM1-5 with CD20.

[0071] Figure 2 It is a graph showing the binding affinity detection results of CD20 antibodies at different concentrations.

[0072] Figure 3 It is a graph showing the EC50 detection results of the F11 original antibody and the humanized antibody HM1-5.

[0073] Figure 4 It is a schematic diagram of the structure of the CAR constructed based on HM5.

[0074] Figure 5 It is composed of Figure 4 The killing ability detection results of the CAR-T cells prepared from the CAR shown against K562-CD20 cells.

[0075] Figure 6 It is a schematic diagram of the structure of the Fmc63-Leu16 CAR.

[0076] Figure 7 It is composed ofFigure 4 Detection results of the killing ability of CAR-T cells prepared from the shown CAR and Fmc63-Leu16 CAR against K562-CD20 cells.

[0077] Figure 8 It is a flow cytometry detection result diagram of CD19 humanized antibody against negative control CHO-K1 cells.

[0078] Figure 9 It is a flow cytometry detection result diagram of CD19 humanized antibody against CHO-K1-CD19 cells.

[0079] Figure 10 It is a detection result diagram of the binding ability of CD19 humanized antibody at different concentrations.

[0080] Figure 11 It is a detection result diagram of the EC50 of CD19 humanized antibody.

[0081] Figure 12 It is a detection result diagram of the affinity of VL2+VH1 and VL3+VH2.

[0082] Figure 13 It is a schematic structural diagram of CAR constructed based on CD19 humanized antibody.

[0083] Figure 14 It is the detection result of the killing ability of CAR-T cells constructed and prepared from VL2+VH1 and VL3+VH2 antibodies against K562-CD19 cells.

[0084] Figure 15 It is a schematic structural diagram of CAR constructed based on bispecific chimeric antigen receptor.

[0085] Figure 16 It is composed of Figure 15 Detection results of the killing ability of CAR-T cells prepared from the shown CAR against K562-CD19 and K562-CD20 cells.

[0086] Figure 17 It is a statistical result diagram of the amplification multiple of cultured cells.

[0087] Figures 18 - 21 It is a flow cytometry detection result diagram of CAR-T cell apoptosis on the 5th / 8th / 10th / 12th day.

[0088] Figure 22 It is a result diagram of the secretion of IL-2 and IFN-γ by CAR-T cells on the 5th, 7th, and 12th days of culture. Specific implementation mode

[0089] The present application will be further described below in conjunction with specific embodiments. However, the protection scope of the present application is not limited thereto. Any person skilled in the art within the technical scope disclosed in the present application, according to the technical solution and inventive concept of the present application, makes equivalent substitutions or changes, and should be covered by the protection scope of the present application.

[0090] The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels without special instructions.

[0091] Recognition and Binding of CD20 Target by Example 1, F11 Antibody and 5 Humanized Antibodies

[0092] Step 1: Screening of CD20 Antibodies and Their Humanization

[0093] An antibody F11 specifically targeting CD20 (single-domain antibody, having CDRs shown in SEQ ID NO.1 - 3, and the full-length amino acid sequence is shown in SEQ ID NO.4) was screened. Further, the F11 antibody was optimized for humanized sequence. The humanization degree scoring system used the T20 score: http: / / abAnalyzer.lakepharma.com for scoring. Five antibodies with scores higher than 85 were selected, and the T20 scores of F11-HM1-HM5 are shown in Table 1.

[0094] Table 1. T20 scores

[0095]

[0096]

[0097] The amino acid sequences of the above-mentioned F11-HM1-HM5 are shown in SEQ ID NO.5 - 9 in sequence. Among them, the sequences of the CDR (Complementarity-determining region, antibody complementary determining region) are the same as those of the original antibody F11, also shown in SEQ ID NO.1 - 3, but the framework region (Framework Residue, FR) has changed relative to the original antibody F11.

[0098] The above-obtained antibody F11 and its humanized antibody were respectively subjected to gene synthesis and subcloned into the pcDNA3.4-IgG1Fc expression vector. After the vector was verified to be correct by sequencing, endotoxin-free plasmid was prepared and then the antibody was expressed. The steps of vector expression are as follows:

[0099] 1) Take out the LVTransm transfection reagent and antibody expression vector from the refrigerator. After thawing at room temperature, pipette up and down to mix well completely. Take out the PBS and warm it to room temperature. Add 4 mL of PBS to one well of a 6-well plate, and add 20 μg of pcDNA3.4-IgG1Fc respectively. After pipetting up and down to mix well, add 60 μL of LVTransm, and immediately pipette up and down to mix evenly. Let it stand at room temperature for 10 minutes.

[0100] 2) Add the above DNA / LVTransm complex to 20 mL of 293F cells, and gently shake to mix well. Place the cells in an incubator at 37 °C and 5% CO2 for further culture.

[0101] 3) After continuous culture for 5 days, centrifuge to collect the culture medium supernatant, filter it with a 0.45 μm filter membrane, collect the supernatant and purify the antibody using a Protein A affinity column. Measure the concentration of the purified antibody and perform SDS-PAGE denaturing electrophoresis at the same time.

[0102] Step 2: Detection of Binding Affinity

[0103] Flow Cytometry Detection Method:

[0104] 1) Resuscitate CHO and CHO-CD20 cells from liquid nitrogen and adjust the cell state to the logarithmic growth phase;

[0105] 2) Divide the cells into several portions, with the number of cells in each portion being 3×10 5 cells. Add 100 μL of antibody expression supernatant or humanized antibody (30 μg / ml, diluted 3-fold for 10 points) respectively. After mixing well, incubate at room temperature for 1 hour;

[0106] 3) Centrifuge at 800 xg at room temperature for 5 minutes, discard the supernatant containing the antibody, and wash the cells 3 times with PBS;

[0107] 4) Add 100 μL of PE-Anti-human IgG (diluted 1:500), mix well, and incubate in the dark at room temperature for 45 min;

[0108] 5) Centrifuge at 800 xg at room temperature for 5 minutes, discard the supernatant containing the secondary antibody, and wash the cells 3 times with PBS;

[0109] 6) Resuspend the cells with 200 μL of PBS for flow cytometry analysis. Prepare CHO and CHO-CD20 cells, incubate with 100 μL of antibody expression supernatant respectively, and then incubate with the PE anti-human IgG fluorescent secondary antibody for flow cytometry detection.

[0110] Furthermore, flow cytometry was performed using the above method, and the results showed that both F11 and its humanized antibodies HM1-HM5 specifically bound to CD20, and the binding affinity of the humanized antibodies HM1-HM5 to CD20 was better than that of the original antibody F11( Figure 1 ).

[0111] The humanized antibodies HM1, 2, and 5 were selected based on the binding strength and degree of humanization, purified, and then reproduced according to the above protocol at different concentrations( Figure 2 ), and the FACS EC50( Figure 3 ) was detected. The results showed that the binding activity of the F11 humanized antibody was consistent with the original one, and the binding of HM2 by flow cytometry was stronger. Therefore, F11-HM2 was selected for further application verification.

[0112] Example 2: Selection of F11 - HM2 for Construction of CAR Vector and Verification of CD20 - Targeted Killing

[0113] Step 1: Construction of Vector

[0114] F11-HM2 (nucleotide sequence shown in SEQ ID NO.10 and amino acid sequence shown in SEQ ID NO.9) was selected to construct a pCDH-EF1a lentiviral expression plasmid containing CAR, and the structure of CAR was as Figure 4 shown. Specifically, the CD8 signal peptide-(F11-HM2)-CD8 hinge region-CD8 transmembrane domain (TM, transmembrane)-41BB co-stimulatory domain-CD3ζ intracellular signaling domain (labeled as CD3z in the figure) were connected in sequence. That is, the amino acid sequence of CAR expressed on the vector was SEQ ID NO.11, 9, 12-15 connected in sequence, and the nucleotide sequence encoding it was SEQ ID NO.16, 10, 17-20 connected in sequence. After the CAR structure, EGFRt was connected through T2A for subsequent fluorescence to reflect the positive rate.

[0115] Step 2: Preparation of Lentivirus

[0116] The lentiviral system plasmids (the above pCDH-EF1α lentiviral expression plasmid, PsPAX2, and pMD2.G three-plasmid system, mixed at a mass ratio of 3:2:1) were transfected into adherent 293T cells in the logarithmic growth phase. The cell culture supernatant harvested 48-72 hours after transfection was concentrated and filtered to obtain CAR lentivirus, which was stored at -80 °C for later use.

[0117] Step 3: Isolation of Peripheral Blood Mononuclear Cells

[0118] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood. T cells were isolated using human CD3 / 28 magnetic beads and virus transduction was performed within 72 hours of activation. After 24 hours of transduction, the culture medium was changed and the cells were cultured until day 8. The cells were collected by centrifugation and resuspended in physiological saline. Flow cytometry was used to identify the EGFRt molecule on the surface of CAR-T cells to ensure that the positive rate was greater than 30%. If the positive rate was lower, EGFRt-PE magnetic beads were used for enrichment sorting.

[0119] Step 4: Detection of Killing Ability

[0120] Killing was performed at an effector-to-target ratio of 5:1, using K562 cells and K562 cells overexpressing CD20 (K562-CD20) as target cells. CAR-T killing was performed for 16 h and detected using LDH.

[0121] The test results are as Figure 5 shown. The results demonstrated that the CAR-T cells constructed with the F11-HM2 sequence had the ability to specifically kill K562 cells overexpressing CD20. In the figure, NCT was the negative control T cells, and B09-HM5 was other CD20 antibodies.

[0122] Example 3: Killing Effect of F11 - HM2 CAR - T is Higher than That of Fmc63 - Leu16 CAR - T

[0123] Step 1: Preparation of CD19 - CD20 Tandem CAR - T Cells (Fmc63 - Leu16 CAR - T)

[0124] Prepare the Bispecific anti-CD20, anti-CD19 CAR T cells (bispecific anti-CD20, anti-CD19 CAR-T cells) disclosed in the article "Bispecific anti-CD20, anti-CD19 CAR T cells for relapsed B cell malignancies: a phase 1 dose escalation and expansion trial". The connection diagram is as Figure 6 shown.

[0125] Step 2: Comparison of Killing Effects

[0126] Compare the CAR-T cells prepared based on F11-HM2 described in Comparative Example 2 with the Fmc63-Leu16 CAR-T prepared above.

[0127] The killing was carried out at an effector-to-target ratio of 1:1, using K562 cells overexpressing CD20 (K562-CD20) as target cells. After 24 hours of CAR-T killing, the results showed that the CAR-T cells constructed with the F11-HM2 sequence had the ability to specifically kill K562 cells overexpressing CD20, and were more effective than Fmc63-Leu16 CAR-T( Figure 7 ).

[0128] Example 4: Humanization of CD19 Antibody Clone fmc63

[0129] Step 1: Humanization of Heavy / Light Chain Variable Regions

[0130] The antibody fmc63 against CD19 (the amino acid sequence of the light chain variable region is shown in SEQ ID NO.21, and the heavy chain variable region is shown in SEQ ID NO.22, where the CDR sequences are marked in Table 2, and its full antibody form is marked as Chimeric in the subsequent result figures, and the single-chain antibody form is marked as Fmc63scfv in the subsequent result figures) was humanized, and the framework sequences with a T20 score higher than 80 were screened according to the T20 scoring system described in Example 1. The T20 scores are shown in Table 3.

[0131] Table 2. Amino acid sequence of fmc63 and CDR markers

[0132]

[0133] Table 3. T20 scores

[0134]

[0135]

[0136] The heavy and light chains of the above-designed humanized antibody were gene-synthesized after codon optimization, and were gene-synthesized separately. The heavy chain was subcloned into the pcDNA3.4-IgG1 expression vector, and the light chain was subcloned into the pcDNA3.4-IgKc expression vector. After the vectors were verified by sequencing, endotoxin-free plasmids were prepared.

[0137] Step 2: Antibody Screening

[0138] The humanized VH and VL antibody expression vectors were combined pairwise, resulting in a total of 15 antibodies, which were named VL1+VH1, VL1+VH2, VL1+VH3, VL1+VH4, VL1+VH5, VL2+VH1, VL2+VH2, VL2+VH3, VL2+VH4, VL2+VH5, VL3+VH1, VL3+VH2, VL3+VH3, VL3+VH4, VL3+VH5, VL4+VH1, VL4+VH2, VL4+VH3, VL4+VH4, VL4+VH5, VL5+VH1, VL5+VH2, VL5+VH3, VL5+VH4, VL5+VH5 respectively. Specifically, the light chain variable region with the amino acid sequence shown in SEQ ID NO.24 can be named VL-HM2, the light chain variable region with the amino acid sequence shown in SEQ ID NO.25 can be named VL-HM3, the heavy chain variable region with the amino acid sequence shown in SEQ ID NO.28 can be named VH-HM1, and the heavy chain variable region with the amino acid sequence shown in SEQ ID NO.29 can be named VH-HM2; then the antibody formed by VL-HM2 and VH-HM1 can be called VL2+VH1 or abbreviated as CD19-21, 21; the antibody formed by VL-HM3 and VH-HM2 can be called VL3+VH2 or abbreviated as CD19-32, 32. The encoding nucleic acids of VL2+VH1 or VL3+VH2 are shown in SEQ ID NO.33 / 34.

[0139] The above 15 antibodies were co-transfected into 293F cells with Chimeric and FMC63scfV for transient expression, and then the binding of the prepared humanized antibodies to the target protein was detected by flow cytometry fluorescence activated cell sorting (FACS). The cells used for detection were CHO-K1( Figure 8 ), CHO-K1-CD19( Figure 9 ). In the figure, the primary antibody was 100 μL supernatant and a positive antibody 10 μg / ml FMC63 scfv, and the secondary antibody was PE anti-human IgG.

[0140] According to Figures 8 - 9 the results, VL2+VH1, VL2+VH2, VL2+VH3, VL3+VH1, VL3+VH2, VL3+VH3 were further detected by FACS EC50( Figure 10 ). According to the FACS EC50 detection, the binding activity of the candidate antibodies was consistent with that of the chimeric antibody, and the average MFI value was statistically analyzed based on the results of flow cytometry detection. The higher the MFI value, the higher the affinity of the corresponding humanized antibody for the antigen CD19( Figure 11 ).

[0141] Step 3: Further Screening

[0142] Further select VL2+VH1 and VL3+VH2 for affinity detection and compare them with the original antibody fmc63. The specific steps are as follows: HIS1K sensor is immobilized with CD19-His (KaiKa Biotech, CD1-HM119) at an immobilization concentration of 5 μg / ml for 350 s. The buffer is PBST (PBS + 0.02% tween20). Chmeric / VL2+VH1 / VL3+VH2 is diluted to 12.5, 6.25, 3.13, 1.56, 0.7813, 0 nM. Affinity detection: Equilibrate for 60 s, bind for 180 s, dissociate for 180 s, and the detection temperature is 25°C. The results are as Figure 12 shown.

[0143] Example 5: Construction of CAR Based on VL2 + VH1 and VL3 + VH2 and Verification of Targeted Killing

[0144] Step 1: Construction of Vector

[0145] For the VL2+VH1 and VL3+VH2 screened in Example 4, express the pCDH-EF1a lentiviral expression plasmid of CAR according to the Figure 12 shown structure. That is, construct according to the CAR structure in step 1 of Example 2, and replace the antibody in step 1 of Example 2 with VL2+VH1 (the coding nucleic acid sequence is as shown in SEQ ID NO.33, where positions 322-366 are the coding nucleic acid of GGGGSGGGGSGGGGS, and GGGGSx3 is used as a linker to connect the heavy chain variable region and the light chain variable region), VL3+VH2 (the coding nucleic acid sequence is as shown in SEQ ID NO.34, where positions 322-366 are the coding nucleic acid of GGGGSGGGGSGGGGS, and GGGGSx3 is used as a linker to connect the heavy chain variable region and the light chain variable region) screened in Example 4.

[0146] Step 2: Preparation of Lentivirus

[0147] Transfect the lentiviral system plasmids (pCDH-EF1α lentiviral expression plasmid, PsPAX2, pMD2.G three-plasmid system, mixed at a mass ratio of 3:2:1) into adherent 293T cells in the logarithmic growth phase. Harvest the cell culture supernatant 48-72 hours after transfection. After concentration and filtration, obtain CAR lentivirus and store it at -80°C for later use. Transduce into peripheral blood mononuclear cells according to the method of Example 2.

[0148] Step 3: Detection of Killing Ability

[0149] The killing was carried out at an effector-to-target ratio of 5:1, using K562 cells and K562 cells overexpressing CD19 (K562-CD19) as target cells, and LDH detection was used to detect the CAR-T killing for 16 h. The results are as Figure 14 It is proved that the CAR-T cells constructed with the VL2+VH1 and VL3+VH2 sequences have the ability to specifically kill K562 cells overexpressing CD19. In the figure, NCT is the negative control T cell, and FMC63-LEU16 is the CD19-CD20 tandem CAR-T (that is, the CD19-CD20 tandem CAR-T cell mentioned in Example 3).

[0150] Example 6: Fmc63 - VL3VH2 Tandem F11 - HM2 CAR - T Targeted Killing of K562 - CD19, K562 - CD20, K562 - CD19 / CD20 Cells:

[0151] Step 1: Construction of Vector Based on Fmc63 - VL3 + VH2 and F11 - HM2 and Preparation of CAR - T Cells

[0152] Select F11-HM2 (the amino acid and nucleic acid sequences are shown in SEQ ID NO.9-10 respectively) screened in Examples 1-3 and VL3+VH2 (the antibody prepared with the amino acid sequences shown in SEQ ID NO.25 and 29 as the light and heavy chain variable regions respectively, and the nucleic acid sequence encoding it is shown in SEQ ID NO.34) screened in Examples 4-5 for tandem connection. The specific connection order of each component of the tandem structure is F11HM2-GGGGSx3-VL3-GGGGSx3-VH2. Use this tandem structure to replace the antibody in step 1 of Example 2 to construct CAR, and the constructed CAR structure is as Figure 15 shown (that is, the bispecific chimeric antigen receptor described in the present application).

[0153] Step 2: Detection of Killing Ability

[0154] Express the plasmid of the CAR in which Fmc63-VL3VH2 and F11-HM2 are tandemly connected constructed in the above steps, prepare lentivirus, and obtain CAR-T cells after infecting cells. Detect its killing ability against K562 cells overexpressing CD19, CD20 or CD19+CD20 (24 h) at an effector-to-target ratio of 5:1.

[0155] Figure 16 The results shown prove that the CAR-T cells constructed with the Fmc63-VL3VH2 tandem F11-HM2 sequence have the ability to specifically kill K562 cells expressing CD19 or CD20 and co-expressing CD19 and CD20. Among them, in the legend, NCT is the control T cell, and 32-F11HM2 is the CAR-T constructed with the Fmc63-VL3VH2 tandem F11-HM2.

[0156] Example 7: Tonic Signaling Detection

[0157] Two selected humanized CD20 single-domain antibodies and the humanized VL3VH2 antibody were respectively used to construct second-generation bispecific CAR lentiviral expression vectors. The second-generation CAR is specifically the CAR consisting of a CD8 signal peptide - antibody - CD8 hinge region - CD8 transmembrane domain - 41BB costimulatory domain - CD3ζ intracellular signaling domain as described in Example 2 above. The antibodies and their legends in the result figures are shown in Table 4.

[0158] Table 4. Antibody names, related SEQ ID NOs. in the CAR, and their labels in the result figures

[0159]

[0160] After constructing the expression plasmids of the above antibodies into CARs, expression was carried out, lentiviruses were prepared, and after infecting cells, CAR-T cells were obtained. The following detections were performed, and the result figures were labeled according to the legends in Table 4.

[0161] 1. Detection of Cell Expansion Multiples

[0162] The CAR-T cells prepared above were cultured and further detected. The specific steps of cell culture were as follows: an appropriate amount of X-Vivo 15 medium (containing 200 IU / mL of IL2, 10 ng / mL of IL7, and 5 ng / mL of IL15) was added, the cells were resuspended with a pipette, and the cell density was adjusted to 0.5 - 0.7×10^6 cells / mL.

[0163] From the 5th day to the 13th day, cell counting was performed every day to count the cell number. The cell number on the initial D0 day was used as the base number to calculate the CAR cell amplification multiple. The results are as Figure 17 shown.

[0164] 2. Detection of Cell Apoptosis

[0165] On the 5th / 8th / 10th / 12th day of culture, some of the cultured CAR-T cells were taken, centrifuged, washed with pre-cooled PBS, resuspended in 300 μL of 1×Binding Buffer, then 5 μL of Annexin V-PE and 5 μL of 7AAD were added for staining. After mixing, they were incubated in the dark for 15 minutes. Blank T cells without any dye were used as the blank group. 400 μL of 1×Binding Buffer was supplemented, and the cells were detected by flow cytometry. The results on the 5th / 8th / 10th / 12th day are successively as Figures 18 - 21 shown.

[0166] 3. Detection of Cytokine Secretion Levels

[0167] On the 5th, 7th, and 12th days respectively, 5×10^5 cells were taken. After being washed twice with PBS, they were resuspended with 1 ml of blank X-Vivo15 medium, placed in an incubator at 37°C with 5% CO2, and cultured for another 24 h. Then, the supernatant was obtained by centrifugation and stored at -80°C. After all the supernatants were collected, the secretion of IL2 and IFN-γ was detected according to the operation instructions of the ELISA kits for IL2 and IFN-γ.

[0168] Results: On D5, D7, and D12 respectively, cells were taken and cultured with blank X-vivo15 for 24 h. Then, the supernatant was taken to detect the secretion of IL2 and IFN-γ. According to the detection results, CAR-T cells hardly secreted IL-2 while secreting IFN-γ, and the secretion increased with the increase of the culture time ( Figure 22 ).

[0169] The results in the above three aspects show that the basal signal (tonic signaling) of the CAR-T cells prepared by concatenating the CD20 antibody and the CD19 antibody screened based on this application is not strong, which can ensure the normal expansion of CAR-T cells and the apoptosis ratio within the normal range. At the same time, it will not release IL-2 under the unactivated condition, and the release level of IFN-γ is controllable.

Claims

1. A CD20 antibody that specifically binds to CD20, the CD20 antibody comprising three complementary determining regions CDR1, CDR2, and CDR3, the amino acid sequence of the complementary determining region CDR1 of the CD20 antibody being as shown in SEQ ID NO.1, the amino acid sequence of the complementary determining region CDR2 being as shown in SEQ ID NO.2, and the amino acid sequence of the complementary determining region CDR3 being as shown in SEQ ID NO.3; The CD20 antibody is a single domain antibody.

2. The CD20 antibody according to claim 1, characterized in that The CD20 antibody is a humanized antibody.

3. The CD20 antibody according to claim 1, characterized in that The amino acid sequence of the CD20 antibody is shown in any one of SEQ ID NOs. 4-9.

4. A fusion protein, wherein the fusion protein is obtained by connecting the CD20 antibody according to any one of claims 1 to 3 and a humanized CD19 antibody in series; The CD19 antibody comprises VH and VL, the amino acid sequence of the VL is shown in SEQ ID NO.25, and the amino acid sequence of the VH is shown in SEQ ID NO.

29.

5. A chimeric antigen receptor, comprising any one of the following: the CD20 antibody according to any one of claims 1 to 3 or the fusion protein according to claim 4.

6. The chimeric antigen receptor according to claim 5, characterized in that The hinge region in the chimeric antigen receptor is selected from the hinge regions of the following molecules: CD8α, CD28, CD3ε, IgG1, and IgG4.

7. The chimeric antigen receptor according to claim 6, characterized in that The hinge region is the CD8α hinge region.

8. The chimeric antigen receptor according to claim 5, characterized in that The transmembrane domain in the chimeric antigen receptor is selected from the transmembrane domains of the following molecules: CD8α, CD28, 4-1BB, CD3ε, IgG1, IgG4, and OX40.

9. The chimeric antigen receptor according to claim 8, characterized in that The transmembrane domain is the CD8α transmembrane domain.

10. The chimeric antigen receptor according to claim 5, characterized in that The co-stimulatory signaling domain in the chimeric antigen receptor is selected from the co-stimulatory signaling domains of the following molecules: 4-1BB, CD28, and OX40.

11. The chimeric antigen receptor according to claim 10, characterized in that The co-stimulatory signaling domain is a 4-1BB co-stimulatory signaling domain.

12. The chimeric antigen receptor according to claim 5, characterized in that The intracellular signaling domain in the chimeric antigen receptor is a CD3ζ intracellular signaling domain.

13. The chimeric antigen receptor according to claim 5, characterized in that The chimeric antigen receptor comprises, from N-terminus to C-terminus: the CD20 antibody according to any one of claims 1 to 3 or the fusion protein according to claim 4 - the CD8α hinge region - the CD8α transmembrane domain - the 4-1BB co-stimulatory signal domain - the CD3ζ intracellular signal transduction domain.

14. A nucleic acid molecule encoding the CD20 antibody according to any one of claims 1 to 3, the fusion protein according to claim 4, or the chimeric antigen receptor according to any one of claims 5 to 13.

15. A recombinant vector comprising the nucleic acid molecule of claim 14.

16. The recombinant vector according to claim 15, characterized in that The recombinant vector is a DNA vector, an RNA vector or a vector derived from a virus.

17. The recombinant vector according to claim 16, characterized in that The DNA vector is a plasmid.

18. The recombinant vector according to claim 16, characterized in that The virus-derived vector is a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a poxvirus vector or a herpesvirus vector.

19. The recombinant vector according to claim 18, characterized in that The retroviral vector is a lentiviral vector.

20. An engineered cell, wherein the engineered cell comprises or expresses one or more of the CD20 antibody of any one of claims 1 to 3, the fusion protein of claim 4, the chimeric antigen receptor of any one of claims 5 to 13, the nucleic acid molecule of claim 14, and the recombinant vector of any one of claims 15 to 19; Alternatively, the engineered cell is an engineered cell prepared by introducing the nucleic acid molecule of claim 14 or the recombinant vector of any one of claims 15 to 19 into the cell.

21. The engineered cell according to claim 20, characterized in that The method of introducing the cells is electroporation, lipofectine transfection or lipofectamin transfection.

22. The engineered cell according to claim 20, characterized in that The cells are of human origin.

23. The engineered cell according to claim 22, characterized in that The cells are immune cells.

24. The engineered cell according to claim 23, characterized in that The immune cells are T cells, B cells, NK cells, myeloid cells or iPS cells.

25. The engineered cell according to claim 24, characterized in that The myeloid cells are monocytes, macrophages or dendritic cells.

26. The engineered cell according to claim 24, characterized in that The T cells are CTL cells, iNKT cells or gdT cells.

27. The engineered cell according to claim 24, characterized in that The immune cells are T cells.

28. The engineered cell according to claim 20, characterized in that The engineered cells are CAR-T cells.

29. A pharmaceutical composition, comprising: the CD20 antibody of any one of claims 1-3, the fusion protein of claim 4, the chimeric antigen receptor of any one of claims 5-13, the nucleic acid molecule of claim 14, the recombinant vector of any one of claims 15-19, and one or more of the engineered cells of any one of claims 20-28.

30. The pharmaceutical composition according to claim 29, characterized in that The pharmaceutical composition also contains pharmaceutically or physiologically acceptable excipients.

31. The pharmaceutical composition according to claim 30, characterized in that The pharmaceutically or physiologically acceptable excipient is a pharmaceutically or physiologically acceptable carrier.

32. The pharmaceutical composition according to claim 30, characterized in that The pharmaceutically or physiologically acceptable excipient is water for injection, sodium chloride solution, glucose solution, a solution containing a surfactant, a pH buffer solution, Ringer's solution or any combination thereof.

33. The pharmaceutical composition according to claim 32, characterized in that The water for injection is bacteriostatic water for injection.

34. An antibody containing a label, wherein the antibody is the CD20 antibody according to any one of claims 1 to 3.

35. The labeled antibody according to claim 34, characterized in that The label is a radionuclide, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, an enzyme and / or a photosensitive diagnostic agent.

36. Use of any one or more of the following products in the preparation of a medicament for treating CD20-related diseases: The CD20 antibody according to any one of claims 1 to 3, the fusion protein according to claim 4, the chimeric antigen receptor according to any one of claims 5 to 13, the nucleic acid molecule according to claim 14, the recombinant vector according to any one of claims 15 to 19, the engineered cell according to any one of claims 20 to 28, and the pharmaceutical composition according to any one of claims 29 to 33; The CD20-related disease is non-Hodgkin's lymphoma, chronic lymphocytic leukemia, chronic myeloid leukemia, multiple sclerosis, rheumatoid arthritis, small lymphocytic lymphoma, mantle cell lymphoma, systemic lupus erythematosus, pemphigus, immune thrombocytopenia or microscopic polyangiitis.

37. The use according to claim 36, characterized in that The systemic lupus erythematosus is lupus nephritis.

38. The use according to claim 36, characterized in that The non-Hodgkin's lymphoma is a B-cell lymphoma.

39. The use according to claim 38, characterized in that The B cell lymphoma is diffuse large B cell lymphoma, follicular lymphoma or marginal zone lymphoma.

40. The use according to claim 36, characterized in that The CD20-related disease is chronic myeloid leukemia.

41. Use of the CD20 antibody according to any one of claims 1 to 3, the fusion protein according to claim 4, or the labeled antibody according to claim 34 or 35 for detecting CD20 for non-diagnostic purposes.

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